In recent years, demand for a protein-immobilized electrode is increasing. For example, in an organism, an electron transfer reaction occurs among a number of protein couples. At the time of examining orientation and the mechanism of electron transfer in formation of a complex among the protein couples, a protein-immobilized electrode is used. In this case, the protein-immobilized electrode is used in such a manner that a protein is immobilized to an electrode and, only when a specific interaction occurs between the protein and another protein, current is detected through the protein-immobilized electrode.
Moreover, in recent years, attention is being paid to use of protein for a photoelectric conversion element. For example, it is taught that photocurrent is obtained from a protein-immobilized electrode in which zinc cytochrome c (which is obtained by substituting iron of horse-heart cytochrome c with zinc) is immobilized to a gold electrode, and a photoelectric conversion element using the protein-immobilized electrode is proposed (refer to patent document 1). However, protein is unstable on the outside of a living organism. Therefore, if long-term stabilization of the photoelectric conversion element is achieved, it is very significant. However, as far as the inventors of the present disclosure know, there is no such a report until now.
Thermusthermophilus-derived cytochrome c552 functions as an electron transfer member in a living organism in a manner similar to the horse-heart cytochrome c. It is known that cytochrome c552 has thermal stability which is much higher than that of horse-heart cytochrome c (refer to non-patent document 1). For example, the denaturation midpoint of general protein is 50 to 60° C. and that of horse-heart cytochrome c is 85° C. On the other hand, the denaturation temperature of cytochrome c552 is immeasurable in a general solution (the upper limit of temperature is 100° C.) and is 100° C. or higher. Moreover, it is reported that the denaturation midpoint of cytochrome c552 in the presence of 4.2M of guanidinium hydrochloride (denaturation agent) is 60 to 70° C.
Because cytochrome c552 has high thermal stability as described above, it is suitable as a device material. Although cytochrome c552 and horse-heart cytochrome c have similar constituent amino acids and similar three-dimensional structures but their environments of an active center heme pocket in which electron transfer is performed are different. Concretely, in the horse-heart cytochrome c, lysine residues having positive charges are dispersed in the entire molecule. In the cytochrome c552, although the number of lysine residues is similar to that in the horse-heart cytochrome c, the lysine residues are not disposed around the heme pocket. It is reported that a complex of cytochrome c552 and its in-vivo redox partner is formed mainly by hydrophobic interaction, according to the structure of the complex (refer to non-patent document 2). Therefore, to immobilize cytochrome c552 to an electrode while maintaining its electron transfer capability, its specific condition search is necessary.
One of known methods for immobilizing horse-heart cytochrome c to an electrode uses a monomolecular film (HS(CH2)10COO—, 1-carboxy-10-decanethiol). Consequently, it is considered to use the immobilization method for the immobilization of cytochrome c552. However, in the method of immobilizing cytochrome c552 to an electrode by using a monomolecular film used in the method for immobilizing horse-heart cytochrome c, an oxidation-reduction current of cytochrome c552 has not been obtained so far.
A French study group reported that they successfully obtained a protein-derived oxidation-reduction current by using a protein-immobilized electrode in which cytochrome c552 is immobilized on a silver electrode (refer to non-patent document 3). However, in a cyclic voltammogram obtained using the protein-immobilized electrode, a peak separation between an oxidation wave and a reduction wave is significant, so that there is a problem in protein orientation control. In addition, silver as the electrode material is susceptible to corrosion and oxidation even in use in a normal environment. That is, because the silver electrode is unsuitable for long-term stable use, it is preferable to use a chemically stable electrode instead of a silver electrode.